DYNAMIC OF SFLT-1 OR ENDOGLiN/PIGF RATIO AS AN INDICATOR FOR IMMINENT PREECLAMPSIA AND/OR HELLP SYNDROME

ABSTRACT

Diagnostic methods and tools relating to diagnosing whether a pregnant subject is at risk for developing preeclampsia and/or early-onset preeclampsia within a short period of time. The methods include determining the amounts of the biomarkers sFlt-1 or Endoglin and PIGF in a first and a second sample of said subject, said first sample being obtained prior to said second sample; calculating a first ratio from the amounts of sFlt-1 or Endoglin and PIGF determined in the first sample, and a second ratio from the amounts of sFlt-1 or Endoglin and PIGF determined in the second sample; and comparing the value of the first and the second ratio, whereby a subject being at risk for developing preeclampsia within a short period of time is diagnosed if the value of the second ratio is increased compared to the value of the first ratio by a factor of at least about 3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/373,098, filed Apr. 2, 2019, which is acontinuation application of U.S. patent application Ser. No. 14/264,377,filed Apr. 29, 2014 (patented as U.S. Pat. No. 10,302,657, issued May28, 2019), which is a continuation of International Application No.PCT/EP2012/072157, filed Nov. 8, 2012, which claims the benefit ofEuropean Patent Application No. 11188422.7, filed Nov. 9, 2011, thedisclosures of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE DISCLOSURE

Pregnancy may be complicated in different ways, it is on one handassociated with pregnancy related mortality of the pregnant woman and,on the other hand, also associated with increased morbidity andmortality of the newborn. Maternal mortality at a rate of 14.5 per100,000 live births, is more frequent in pregnant women above the age of39 years and may be caused by haemorrhage, thrombotic pulmonaryembolism, infections, cardiomyopathy and cardiovascular andnoncardiovascular conditions as well as hypertensive disorders amongwhich preeclampsia is frequent (Berg 2010, Obstretics and Gynecology:116: 1302-1309).

Preeclampsia complicates approximately 2 to 8 percent of all pregnanciesand is a major contributor to maternal and fetal mortality worldwide(Duley 2009, Semin Perinatol: 33: 130-37). Preeclampsia is generallydefined as pregnancy associated or induced hypertension. It ischaracterized by hypertension and proteinuria. Hypertension is definedin this context as blood pressure of 140 mmHg (systolic) to 90 mmHg(diastolic) or more at two independent measurements, wherein said twomeasurements have been made at least 6 hours apart. Proteinuria isindicated by 300 mg/dL protein or more in a 24-hour urine sample.However, the definitions of preeclampsia are subject to debate and candiffer among societies.

SUMMARY OF THE DISCLOSURE

The instant disclosure provides a reliable assay for identifyingapparently healthy pregnant females that are at risk of developingimminent preeclampsia and, in particular, imminentearly-onset-preeclampsia

Embodiments of the invention are concerned with diagnostic methods andtools. Specifically, the present invention relates to a method fordiagnosing whether a pregnant subject is at risk for developingpreeclampsia within a short period of time comprising: (a) determiningthe amounts of the biomarkers sFlt-1 or Endoglin and PIGF in a first anda second sample of said subject, wherein said first sample has beenobtained prior to said second sample, (b) calculating a first ratio fromsaid amounts of sFlt-1 or Endoglin and PIGF determined in the firstsample and a second ratio from said amounts of sFlt-1 or Endoglin andPIGF determined in the second sample and (c) comparing the value of thesaid first and the said second ratio, whereby a subject being at riskfor developing preeclampsia within a short period of time is diagnosedif the value of the second ratio is increased compared to the value ofthe first ratio by a factor of at least about 3. The present inventionfurther relates to a method for differentiating between a pregnantsubject being at risk of developing early-onset-preeclampsia and apregnant subject being not at risk of developingearly-onset-preeclampsia. Moreover, encompassed by the invention aredevices and kits for carrying out these methods. The present inventionalso relates to a system for performing an optimized risk assessment ofdeveloping preeclampsia as disclosed herein and to reagents and kitsused in performing the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of this disclosure, and the manner of attaining them, willbecome more apparent and the disclosure itself will be better understoodby reference to the following description of embodiments of thedisclosure taken in conjunction with the accompanying drawing.

FIG. 1A shows a graph presenting the distribution of the week ofgestation for individual subjects in the study for the group ofpreeclampsia (PE) outcome patients and the healthy controls. The firstvisit is shown in the box plots.

FIG. 1B shows another graph presenting the distribution of the week ofgestation for individual subjects in the study for the group ofpreeclampsia (PE) outcome patients and the healthy controls. The secondvisit is shown in the box plots.

FIG. 1C shows a graph presenting the distribution of the week ofgestation for individual subjects in the study for the group ofpreeclampsia (PE) outcome patients and the healthy controls. The timedifference in days between the visits is shown in the box plots.

FIG. 2A shows sFlt-1/PIGF ratios for the PE group and the healthycontrols at different visits in normal and log scales.

FIG. 2B shows another graph presenting sFlt-1/PIGF ratios for the PEgroup and the healthy controls at different visits in normal and logscales.

FIG. 3A shows differences of sFlt-1/PIGF ratios compared to gestationalage.

FIG. 3B presents another graph showing differences of sFlt-1/PIGF ratioscompared to the time point of measurement.

FIG. 4A shows a time to diagnosis of PE/HELLP versus both values ofsFlt-1/PIGF ratio plot (left).

FIG. 4B shows the slopes between the sFlt-1/PIGF ratios at first andsecond visit from patients of the PE/HELLP group.

FIG. 5A shows sFlt-1/PIGF ratios at different weeks of gestation.

FIG. 5B shows Endoglin (sEng)/PIGF ratios at different weeks ofgestation.

FIG. 6A shows sEng/PIGF ratios for the PE group and the healthy controlsat different visits in normal scales.

FIG. 6B shows sEng/PIGF ratios for the PE group and the healthy controlsat different visits in log scales.

FIG. 7A shows differences of sEng/PIGF ratios compared to gestationalage.

FIG. 7B shows differences of sEng/PIGF ratios compared to the time pointof measurement.

FIG. 8A shows a time to diagnosis of PE/HELLP versus values of sEng/PIGFratio plot.

FIG. 8B shows the slopes between the sEng/PIGF ratios at first andsecond visit from patients of the PE/HELLP group.

Corresponding reference characters indicate corresponding partsthroughout the several views. Although the drawings representembodiments of the present disclosure, the drawings are not necessarilyto scale and certain features may be exaggerated in order to betterillustrate and explain the present disclosure. The exemplifications setout herein illustrate an exemplary embodiment of the disclosure, in oneform, and such exemplifications are not to be construed as limiting thescope of the disclosure in any manner.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE DISCLOSURE

The embodiments disclosed herein are not intended to be exhaustive orlimit the disclosure to the precise form disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

Preeclampsia complicates approximately 2 to 8 percent of all pregnanciesand is a major contributor to maternal and fetal mortality worldwide(Duley 2009, Semin Perinatol: 33: 130-37). Preeclampsia is generallydefined as pregnancy associated or induced hypertension. It ischaracterized by hypertension and proteinuria. Hypertension is definedin this context as blood pressure of 140 mmHg (systolic) to 90 mmHg(diastolic) or more at two independent measurements, wherein said twomeasurements have been made at least 6 hours apart. Proteinuria isindicated by 300 mg/dL protein or more in a 24-hour urine sample.However, the definitions of preeclampsia are subject to debate and candiffer among societies. Details are also found in the standard textbooks of medicine and the Guidelines of the various clinical societies,e.g., ACOG Practice Bulletin, Clinical Management Guidelines forObstetrician—Gynecologists, no.: 33, January 2002 or Leitlinien,Empfehlungen, Stellungnahmen of the Deutschen Gesellschaft fOrGynakologie and Geburtshilfe e.V., August 2008.

The pathogenesis of preeclampsia is largely unknown. It is believed,however, to be caused by disturbed placental function associated withimpaired remodeling of the spiral artery. Flow defects occurring in theprocess of the development of preeclampsia are associated with ischemiathat ultimately results in the release of anti-angiogenic factors intothe circulations such as sFlt-1 and Endoglin.

The sole treatment of preeclampsia until today is the termination ofpregnancy either by premature vaginal or caesarean delivery. Asdiscussed above maternal risks and fetal viability are significantlyimpaired in case of preeclampsia before gestational week 34.Accordingly, attempts should be made to delay delivery and to therebyimprove survival of the newborn.

The early and reliable diagnosis of preeclampsia and, in particular, theearly onset type of preeclampsia which occurs as early as 20 to 34 weeksof gestation is decisive for clinical management of the disease. It willbe understood that pregnant females suffering from preeclampsia needspecial care such as close monitoring, supportive therapeutic measuresand, in the case of progression into severe preeclampsia,hospitalization in specialized hospitals having maternal fetal intensivecare units (MFICUs). In particular, the early-onset-preeclampsia ischallenging for the clinicians in light of the severe side-effects andthe usual adverse outcomes associated therewith. Moreover, the early andreliable diagnosis of preeclampsia as well as the prediction ofpreeclampsia is decisive for the planning of preventive or therapeuticintervention studies (Ohkuchi 2011, Hypertension 58: 859-866).

Accordingly, embodiments of the present invention relate to a method fordiagnosing whether a pregnant subject is at risk for developingpreeclampsia within a short period of time comprising: a) determiningthe amounts of the biomarkers sFlt-1 or Endoglin and PIGF in a first anda second sample of said subject, wherein said first sample has beenobtained prior to said second sample; b) calculating a first ratio fromsaid amounts of sFlt-1 or Endoglin and PIGF determined in the firstsample and a second ratio from said amounts of sFlt-1 or Endoglin andPIGF determined in the second sample; c) comparing the value of the saidfirst and the said second ratio, whereby a subject being at risk fordeveloping preeclampsia within a short period of time is diagnosed ifthe value of the second ratio is increased compared to the value of thefirst ratio by a factor of at least about 3.

The method of the present invention may comprise an ex vivo method.Moreover, it may comprise steps in addition to those explicitlymentioned above. For example, further steps may relate to samplepre-treatments or evaluation of the results obtained by the method. Themethod may be carried out manually or assisted by automation. Forexample, step (a), (b) and/or (c) may in total or in part be assisted byautomation, e.g., by a suitable robotic and sensory equipment for thedetermination in step (a), a computer-implemented calculation algorithmon a data processing device in step (b) or comparison and/or diagnosisalgorithm on a data processing device in step (c).

Accordingly, the present invention also relates to a system foroptimizing a risk assessment based on a clinical prediction rule forclassifying pregnant subjects, comprising a) an analyzer unit configuredto contact, in vitro, a portion of a second sample from a pregnantsubject with a ligand comprising specific binding affinity for sFlt-1and/or Endoglin and configured to contact, in vitro, a portion of asample from a pregnant subject with a ligand comprising specific bindingaffinity for PIGF; b) an analyzer unit configured to detect a signalfrom the portions of the sample from the subject contacted with theligands, c) a computing device having a processor and in operablecommunication with said analysis units, and d) a non-transient machinereadable media including a plurality of instruction executable by aprocessor, the instructions, when executed, calculating an amount ofsFlt-1 and/or Endoglin, calculating an amount of PIGF, calculating asecond ratio from said amounts of sFlt-1 or Endoglin and PIGF determinedin the sample and comparing the ratio thus calculated with a first ratioobtained from a first sample, thereby optimizing the risk assessmentbased on the clinical prediction rule for classifying pregnant subjects.

The term “preeclampsia” as used herein refers to a medical conditionwhich is characterized by hypertension and proteinuria. Preeclampsiaoccurs in pregnant female subjects and the hypertension is also referredto as pregnancy-induced hypertension. The pregnancy-induced hypertensionmay be identified to be present in a subject by two blood pressuremeasurements of 140 mmHg (systolic) to 90 mmHg (diastolic) or more,wherein said two measurements have been made at least 6 hours apart.Proteinuria may be identified to be present by 300 mg/dL protein or morein a 24-hour urine sample. Preeclampsia may progress to eclampsia, alife-threatening disorder characterized by the appearance oftonic-clonic seizures or coma conditions. Symptoms associated withsevere preeclampsia are oligouria of less than 500 ml within 24 hours,cerebral or visual disturbance, pulmonary edema or cyanosis, epigastric-or right upper quadrant- pain, impaired liver function,thrombocytopenia, fetal growth restriction. Subjects suffering frompreeclampsia with hepatic involvement may further develop the HELLPsyndrome. Accordingly, a subject according to the invention which is atrisk of developing preeclampsia, may also potentially be at risk ofdeveloping the HELLP syndrome. The HELLP syndrome is associated with ahigh risk of adverse outcomes such as placental abruption, renalfailure, subcapsular hepatic hematoma, recurrent preeclampsia, pretermdelivery, or even materal and/or fetal death. Further details ofpreeclampsia and the accompanying symptoms as well as the follow updiseases such as HELLP syndrome or eclampsia can be found, for example,in standard text books of medicine or Guidelines of the relevant medicalsocieties. Details can be found, e.g., in ACOG Practice Bulletin,Clinical Management Guidelines for Obstetrician—Gynecologists, no.: 33,January 2002 or Leitlinien, Empfehlungen, Stellungnahmen of theDeutschen Gesellschaft fOr Gynakologie and Geburtshilfe e.V., August2008. Preeclampsia occurs in up to 10% of pregnancies usually in thesecond or third trimester. However, some females develop preeclampsia asearly as in week 20 of gestation.

Within week 20 to 34 of gestation, preeclampsia is also calledearly-onset-preeclampsia while preeclampsia which occurs after week 34of gestation is also termed late-onset-preeclampsia. It will beunderstood that the early-onset-preeclampsia, usually, is accompanied bymore severe side-effects and adverse outcomes compared to the usuallyrelatively mild late-onset-preeclampsia. The phrase “at risk fordeveloping preeclampsia” refers to a pregnant subject which will developpreeclampsia within a prognostic time window in the future with astatistically significantly increased likelihood compared to a pregnantsubject which is not at risk for developing preeclampsia. For example,that likelihood may be at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99% or up to 100%.Further details on statistics are found elsewhere herein.

The term “subject” as used herein relates to animals, for examplemammals, and humans. The subject according to the present invention is apregnant subject, i.e. a pregnant female. According to variousembodiments, the subject according to the present invention exhibits nosymptoms of preeclampsia. Such symptoms of preeclampsia include clinicalsymptoms of preeclampsia as specified elsewhere herein. The symptoms maycomprise at least one symptom selected from the group consisting of:epigastric pain, headache, visual disturbance, and edema. However, thesubject according to the present invention may also exhibit at least oneof the aforementioned symptoms and may, thus, be suspected to sufferfrom preeclampsia already.

In some embodiments, the pregnant subject according to the presentinvention is between about week 15 and about week 34 of gestation, forexample between about week 15 and about week 30 of gestation.

According to instant disclosure, the method of the present invention canbe used in routine screening approaches of apparently healthy pregnantsubjects. However, the pregnant subject envisaged by the presentinvention may also belong into a risk group having a higher prevalencefor preeclampsia. Pregnant subjects suffering from adiposity,hypertension, autoimmune diseases such as Lupus erythematosus,thrombophilias or diabetes mellitus have an increased prevalence fordeveloping preeclampsia in general. The same applies for subjects thatsuffered from preeclampsia, eclampsia and/or HELLP syndrome in aprevious pregnancy. Furthermore, elderly females who are pregnant forthe first time do also exhibit a predisposition for developingpreeclampsia. The likelihood for developing preeclampsia, however, isdecreasing with the number of pregnancies.

The term “diagnosing” as used herein means assessing whether a subjectis at risk of developing preeclampsia within a short period of time, ornot. In some embodiments, said short period of time is a period of timeless than 4 weeks, or between about 2 to about 3 weeks, and, in somecases, a time period of about 16 days. As will be understood by thoseskilled in the art, such an assessment is usually not intended to becorrect for 100% of the subjects to be diagnosed. The term, however,requires that the assessment is correct for a statistically significantportion of the subjects (e.g., a cohort in a cohort study). Whether aportion is statistically significant can be determined without furtherado by the person skilled in the art using various well known statisticevaluation tools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test etc. Details arefound, for example, in Dowdy and Wearden, Statistics for Research, JohnWiley & Sons, New York 1983. Exemplary conditions of confidenceintervals according to the instant disclosure are at least 90%, at least95%, at least 97%, at least 98% or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.

The term “sample” refers to a sample of a body fluid, to a sample ofseparated cells or to a sample from a tissue or an organ. Samples ofbody fluids can be obtained by well-known techniques and include,samples of blood, plasma, serum, or urine. Tissue or organ samples maybe obtained from any tissue or organ by, e.g., biopsy. Separated cellsmay be obtained from the body fluids or the tissues or organs byseparating techniques such as centrifugation or cell sorting. Cell-,tissue- or organ samples are obtained from those cells, tissues ororgans which express or produce the peptides referred to herein.

The “first sample” according to the method of the present invention hasbeen obtained prior to the “second sample”. It will be understood thatthe first sample and the second sample are samples of the same type ofsample material, i.e., are from the same type of body fluid, cells,tissue or organ. Moreover, the first sample has been prior to the secondsample, for example, at two subsequent regular medical investigationsduring pregnancy wherein the first sample has been taken at the earlierinvestigation and the second sample has been taken at the laterinvestigation.

According to some embodiments, the first sample has been obtained about1 week to about 15 weeks, about 2 weeks to about 6 weeks and, in somecases about 4 to about 5 weeks prior to the said second sample.

The term “sFlt-1” as used herein refers to polypeptide which is asoluble form of the fms-like tyrosine kinase 1. The polypeptide is alsoreferred to as soluble VEGF receptor 1 (sVEGF R1) in the art (see, e.g.,Sunderji 2010, Am J Obstet Gynecol 202: 40e1-7). It was identified inconditioned culture medium of human umbilical vein endothelial cells.The endogenous sFlt1 receptor is chromatographically and immunologicallysimilar to recombinant human sFlt1 and binds [1251] VEGF with acomparable high affinity. Human sFlt1 is shown to form a VEGF-stabilizedcomplex with the extracellular domain of KDR/Flk-1 in vitro. Accordingto the instant disclosure, sFlt1 refers to human sFlt1 as describe inKendall 1996, Biochem Biophs Res Commun 226(2): 324-328; for amino acidsequences, see, e.g., also Genebank accession numbers P17948, Cl: 125361for human and BAA24499.1, Cl: 2809071 for mouse sFlt-1 (Genebank isavailable from the NCBI, USA under ncbi.nlm.nih.gov/entrez). The termalso encompasses variants of the aforementioned human sFlt-1polypeptides. Such variants have at least the same essential biologicaland immunological properties as the aforementioned sFlt-1 polypeptide.In particular, they share the same essential biological andimmunological properties if they are detectable by the same specificassays referred to in this specification, e.g., by ELISA assays usingpolyclonal or monoclonal antibodies specifically recognizing the saidsFlt-1 polypeptides. Moreover, it is to be understood that a variant asreferred to in accordance with the present invention shall have an aminoacid sequence which differs due to at least one amino acid substitution,deletion and/or addition wherein the amino acid sequence of the variantis still, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% identical with the amino sequence of the specificsFlt-1 polypeptide, in some cases over the entire length of the humansFlt-1, respectively. The degree of identity between two amino acidsequences can be determined by algorithms well known in the art.According to some embodiments, the degree of identity is to bedetermined by comparing two optimally aligned sequences over acomparison window, where the fragment of amino acid sequence in thecomparison window may comprise additions or deletions (e.g., gaps oroverhangs) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment. The percentageis calculated by determining the number of positions at which theidentical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison may be conductedby the local homology algorithm disclosed by Smith 1981, Add. APL. Math.2:482, by the homology alignment algorithm of Needleman 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson 1988, Proc.Natl. Acad Sci. (USA) 85: 2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis.), or by visual inspection. Given that twosequences have been identified for comparison, GAP and BESTFIT areemployed to determine their optimal alignment and, thus, the degree ofidentity. According to some embodiments, the default values of 5.00 forgap weight and 0.30 for gap weight length are used. Variants referred toabove may be allelic variants or any other species specific homologs,paralogs, or orthologs. Variants referred to above may be allelicvariants or any other species specific homologs, paralogs, or orthologs.Moreover, the variants referred to herein include fragments or subunitsof the specific sFlt-1 polypeptides or the aforementioned types ofvariants as long as these fragments have the essential immunological andbiological properties as referred to above. Such fragments may be, e.g.,degradation products of the sFlt-1 polypeptides. Variants are deemed toshare the same essential biological and immunological properties if theyare detectable by the same specific assays referred to in thisspecification, e.g., by ELISA assays using polyclonal or monoclonalantibodies specifically recognizing the said sFlt-1 polypeptides. Anillustrative assay is described in the accompanying Examples. Furtherincluded are variants which differ due to posttranslationalmodifications such as phosphorylation or myristylation. sFlt-1 may bedetected in bound or free form or as total sFlt-1 amount in a sample.

The term “Endoglin” as used herein refers to a polypeptide having amolecular weight of 180 kDa non-reduced, 95 kDa after reduction and 66kDa in its reduced and N-deglycosylated form. The polypeptide is capableof forming dimers and binds to TGF-13 and TGF-13 receptors. Also,Endoglin refers to human Endoglin. According to embodiments of theinstant disclosure, human Endoglin has an amino acid sequence as shownin Genebank accession number AAC63386.1, GI: 3201489. Two Endoglinisoforms, S-Endoglin and L-Endoglin have been described. L-Endoglinconsists of total of 633 amino acids with a cytoplasmic tail of 47 aminoacids while S-Endoglin consists of 600 amino acids with a cytoplasmictail of 14 amino acids. According to some embodiments, Endoglin as usedherein is soluble Endoglin (sEng). Soluble Endoglin as referred toherein is preferably described in EP 1 804 836 B 1. Moreover, it is tobe understood that a variant as referred to in accordance with thepresent invention may have an amino acid sequence which differs due toat least one amino acid substitution, deletion and/or addition whereinthe amino acid sequence of the variant is still, preferably, at least50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical withthe amino sequence of the specific Endoglin. Variants may be allelicvariants, splice variants or any other species specific homologs,paralogs, or orthologs. Moreover, the variants referred to hereininclude fragments of the specific Endoglin or the aforementioned typesof variants as long as these fragments have the essential immunologicaland biological properties as referred to above. Such fragments may be,e.g., degradation products of Endoglin. Variants are deemed to share thesame essential biological and immunological properties if they aredetectable by the same specific assays referred to in thisspecification, e.g., by ELISA assays using polyclonal or monoclonalantibodies specifically recognizing the said Endoglin polypeptides. Anexemplary assay is described in the accompanying Examples. Furtherincluded are variants which differ due to posttranslationalmodifications such as phosphorylation or myristylation. Endoglin may bedetected in bound or free form or as total Endoglin amount in a sample.

The term “PIGF (Placental Growth Factor)” as used herein refers to aplacenta derived growth factor which is a polypeptide having 149 aminoacids in length and being highly homologous to the platelet-derivedgrowth factor-like region of human vascular endothelial growth factor(VEGF). Like VEGF, PIGF has angiogenic activity in vitro and in vivo.For example, biochemical and functional characterization of PIGF derivedfrom transfected COS-1 cells revealed that it is a glycosylated dimericsecreted protein which is able to stimulate endothelial cell growth invitro (Maqlione 1993, Oncogene 8(4):925-31). According to embodiments ofthe instant disclosure, PIGF refers to human PIGF, for example, to humanPIGF having an amino acid sequence as shown in Genebank accession numberP49763, GI: 17380553. The term encompasses variants of said specifichuman PIGF. Such variants have at least the same essential biologicaland immunological properties as the specific PIGF polypeptide. Variantsare deemed to share the same essential biological and immunologicalproperties if they are detectable by the same specific assays referredto in this specification, e.g., by ELISA assays using polyclonal ormonoclonal antibodies specifically recognizing the said PIGFpolypeptides. An exemplary assay is described in the accompanyingExamples. Moreover, it is to be understood that a variant as referred toin accordance with the present invention shall have an amino acidsequence which differs due to at least one amino acid substitution,deletion and/or addition wherein the amino acid sequence of the variantis still, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% identical with the amino sequence of the specific PIGFpolypeptides. The degree of identity between two amino acid sequencescan be determined by algorithms well known in the art and describedelsewhere herein. Variants referred to above may be allelic variants orany other species specific homologs, paralogs, or orthologs. Moreover,the variants referred to herein include fragments of the specific PLGFpolypeptides or the aforementioned types of variants as long as thesefragments have the essential immunological and biological properties asreferred to above. Such fragments may be, e.g., degradation products orsplice variants of the PLGF polypeptides. Further included are variantswhich differ due to posttranslational modifications such asphosphorylation or myristylation. PIGF may be detected in bound or freeform or as total PIGF amount in a sample.

Determining the amount of any peptide or polypeptide referred to in thisspecification relates to measuring the amount or concentration, forexample, semi-quantitatively or quantitatively. Measuring can be donedirectly or indirectly. Direct measuring relates to measuring the amountor concentration of the peptide or polypeptide based on a signal whichis obtained from the peptide or polypeptide itself and the intensity ofwhich directly correlates with the number of molecules of the peptidepresent in the sample. Such a signal—sometimes referred to herein asintensity signal—may be obtained, e.g., by measuring an intensity valueof a specific physical or chemical property of the peptide orpolypeptide. Indirect measuring includes measuring of a signal obtainedfrom a secondary component (i.e., a component not being the peptide orpolypeptide itself) or a biological read out system, e.g., measurablecellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present invention, determining the amount of apeptide or polypeptide can be achieved by all known means fordetermining the amount of a peptide in a sample. Said means compriseimmunoassay devices and methods which may utilize labeled molecules invarious sandwich, competition, or other assay formats. Said assays willdevelop a signal which is indicative for the presence or absence of thepeptide or polypeptide. Moreover, the signal strength can, for example,be correlated directly or indirectly (e.g. reverse- proportional) to theamount of polypeptide present in a sample. Further suitable methodscomprise measuring a physical or chemical property specific for thepeptide or polypeptide such as its precise molecular mass or NMRspectrum. Said methods comprise, in some example embodiments,biosensors, optical devices coupled to immunoassays, biochips,analytical devices such as mass-spectrometers, NMR-analysers, orchromatography devices. Further, methods include micro-plate ELISA-basedmethods, fully-automated or robotic immunoassays (available for exampleon ELECSYS™ analyzers), CBA (an enzymatic Cobalt Binding Assay,available, for example, on ROCHE-HITACHI™ analyzers), and latexagglutination assays (available, for example, on ROCHE-HITACHI™analyzers).

According to embodiments of the instant disclosure, determining theamount of a peptide or polypeptide comprises the steps of (a) contactinga cell capable of eliciting a cellular response the intensity of whichis indicative of the amount of the peptide or polypeptide with the saidpeptide or polypeptide for an adequate period of time, (b) measuring thecellular response. For measuring cellular responses, the sample orprocessed sample is, for example, added to a cell culture and aninternal or external cellular response is measured. The cellularresponse may include the measurable expression of a reporter gene or thesecretion of a substance, e.g., a peptide, polypeptide, or a smallmolecule. The expression or substance shall generate an intensity signalwhich correlates to the amount of the peptide or polypeptide. Accordingto a illustrative embodiments, said steps of contacting, removing andmeasuring may be performed by an analyser unit of the system disclosedherein. According to some embodiments, said steps may be performed by asingle analyzer unit of said system or by more than one analyser unit inoperable communication with each other. For example, according to aspecific embodiment, said system disclosed herein may include a firstanalyser unit for performing said steps of contacting and removing and asecond analyser unit, operably connected to said first analyzer unit bya transport unit (for example, a robotic arm), which performs said stepof measuring.

Also, according to embodiments of the instant disclosure, determiningthe amount of a peptide or polypeptide comprises the step of measuring aspecific intensity signal obtainable from the peptide or polypeptide inthe sample. As described above, such a signal may be the signalintensity observed at an m/z variable specific for the peptide orpolypeptide observed in mass spectra or a NMR spectrum specific for thepeptide or polypeptide.

Determining the amount of a peptide or polypeptide may, for example,comprise the steps of (a) contacting the peptide with a specific ligand,(b) removing non-bound ligand, (c) measuring the amount of bound ligand.The bound ligand will generate an intensity signal. Binding according tothe present invention includes both covalent and non-covalent binding. Aligand according to the present invention can be any compound, e.g., apeptide, polypeptide, nucleic acid, or small molecule, binding to thepeptide or polypeptide described herein. Exemplary ligands includeantibodies, nucleic acids, peptides or polypeptides such as receptors orbinding partners for the peptide or polypeptide and fragments thereofcomprising the binding domains for the peptides, and aptamers, e.g.,nucleic acid or peptide aptamers. Methods to prepare such ligands arewell-known in the art. For example, identification and production ofsuitable antibodies or aptamers is also offered by commercial suppliers.The person skilled in the art is familiar with methods to developderivatives of such ligands with higher affinity or specificity. Forexample, random mutations can be introduced into the nucleic acids,peptides or polypeptides. These derivatives can then be tested forbinding according to screening procedures known in the art, e.g., phagedisplay. Antibodies as referred to herein include both polyclonal andmonoclonal antibodies, as well as fragments thereof, such as Fv, Fab andF(ab)2 fragments that are capable of binding antigen or hapten. Thepresent invention also includes single chain antibodies and humanizedhybrid antibodies wherein amino acid sequences of a non-human donorantibody exhibiting a desired antigen-specificity are combined withsequences of a human acceptor antibody. The donor sequences will usuallyinclude at least the antigen-binding amino acid residues of the donorbut may comprise other structurally and/or functionally relevant aminoacid residues of the donor antibody as well. Such hybrids can beprepared by several methods well known in the art. According toembodiments of the instant disclosure, the ligand or agent bindsspecifically to the peptide or polypeptide. Specific binding accordingto the present invention means that the ligand or agent should not bindsubstantially to, i.e., cross-react with, another peptide, polypeptideor substance present in the sample to be analyzed. According toembodiments of the instant disclosure, the specifically bound peptide orpolypeptide should be bound with at least 3 times higher, more forexample, at least 10 times higher and even more for example at least 50times higher affinity than any other relevant peptide or polypeptide.Non-specific binding may be tolerable, if it can still be distinguishedand measured unequivocally, e.g., according to its size on a WesternBlot, or by its relatively higher abundance in the sample. Binding ofthe ligand can be measured by any method known in the art. According toembodiments of the instant disclosure, said method is semi-quantitativeor quantitative. Further suitable techniques for the determination of apolypeptide or peptide are described in the following.

Also, binding of a ligand may be measured directly, e.g., by NMR orsurface plasmon resonance. Measurement of the binding of a ligand,according to some embodiments, is performed by an analyzer unit of asystem disclosed herein. Thereafter, an amount of the measured bindingmay be calculated by a computing device of a system disclosed herein.Second, if the ligand also serves as a substrate of an enzymaticactivity of the peptide or polypeptide of interest, an enzymaticreaction product may be measured (e.g. the amount of a protease can bemeasured by measuring the amount of cleaved substrate, e.g., on aWestern Blot). Alternatively, the ligand may exhibit enzymaticproperties itself and the “ligand/peptide or polypeptide” complex or theligand which was bound by the peptide or polypeptide, respectively, maybe contacted with a suitable substrate allowing detection by thegeneration of an intensity signal. For measurement of enzymatic reactionproducts, in some illustrative cases the amount of substrate issaturating. The substrate may also be labeled with a detectable labelprior to the reaction. According to embodiments of the instantdisclosure, the sample is contacted with the substrate for an adequateperiod of time. An adequate period of time refers to the time necessaryfor a detectable, in some cases, amount of product to be produced.Instead of measuring the amount of product, the time necessary forappearance of a given (e.g., detectable) amount of product can bemeasured. Also, the ligand may be coupled covalently or non-covalentlyto a label allowing detection and measurement of the ligand.

Labeling may be done by direct or indirect methods. Direct labelinginvolves coupling of the label directly (covalently or non-covalently)to the ligand. Indirect labeling involves binding (covalently ornon-covalently) of a secondary ligand to the first ligand. The secondaryligand should specifically bind to the first ligand. Said secondaryligand may be coupled with a suitable label and/or be the target(receptor) of tertiary ligand binding to the secondary ligand. The useof secondary, tertiary or even higher order ligands is often used toincrease the signal. Suitable secondary and higher order ligands mayinclude antibodies, secondary antibodies, and the well-knownstreptavidin-biotin system (Vector Laboratories, Inc.).

The ligand or substrate may also be “tagged” with one or more tags asknown in the art. Such tags may then be targets for higher orderligands. Suitable tags include biotin, digoxygenin, His-Tag,Glutathion-S-Transferase, FLAG, GFP, myc-tag, influenza A virushaemagglutinin (HA), maltose binding protein, and the like. In the caseof a peptide or polypeptide, the tag may be at the N-terminus and/orC-terminus.

Suitable labels are any labels detectable by an appropriate detectionmethod. Typical labels include gold particles, latex beads, acridanester, luminol, ruthenium, enzymatically active labels, radioactivelabels, magnetic labels (“e.g. magnetic beads”, including paramagneticand superparamagnetic labels), and fluorescent labels. Enzymaticallyactive labels include e.g. horseradish peroxidase, alkaline phosphatase,beta-Galactosidase, Luciferase, and derivatives thereof. Suitablesubstrates for detection include di-amino-benzidine (DAB),3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazoliumchloride and 5-bromo-4-chloro-3-indolyl-phosphate, available asready-made stock solution from Roche Diagnostics), CDP-Star™ (AmershamBiosciences), ECF™ (Amersham Biosciences).

A suitable enzyme-substrate combination may result in a colored reactionproduct, fluorescence or chemoluminescence, which can be measuredaccording to methods known in the art (e.g. using a light-sensitive filmor a suitable camera system). As for measuring the enyzmatic reaction,the criteria given above apply analogously. Typical fluorescent labelsinclude fluorescent proteins (such as GFP and its derivatives), Cy3,Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568).Further fluorescent labels are available e.g. from Molecular Probes(Oregon). Also the use of quantum dots as fluorescent labels iscontemplated. Typical radioactive labels include ³⁵S, ¹²⁵I, ³²V, ³³P andthe like. A radioactive label can be detected by any method known andappropriate, e.g. a light-sensitive film or a phosphor imager. Suitablemeasurement methods according the present invention also includeprecipitation (particularly immunoprecipitation),electrochemiluminescence (electro-generated chemiluminescence), RIA(radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwichenzyme immune tests, electrochemiluminescence sandwich immunoassays(ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA),scintillation proximity assay (SPA), turbidimetry, nephelometry,latex-enhanced turbidimetry or nephelometry, or solid phase immunetests. Further methods known in the art (such as gel electrophoresis, 2Dgel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE),Western Blotting, and mass spectrometry), can be used alone or incombination with labeling or other detection methods as described above.

The amount of a peptide or polypeptide may be, for example, determinedas follows: (a) contacting a solid support comprising a ligand for thepeptide or polypeptide as specified above with a sample comprising thepeptide or polypeptide, (b) removing unbound peptide or polypeptide aswell as remaining sample material and (c) measuring the amount peptideor polypeptide which is bound to the support. The ligand may be chosenfrom the group consisting of nucleic acids, peptides, polypeptides,antibodies and aptamers and is, for example, present on a solid supportin immobilized form. Materials for manufacturing solid supports are wellknown in the art and include, inter alia, commercially available columnmaterials, polystyrene beads, latex beads, magnetic beads, colloid metalparticles, glass and/or silicon chips and surfaces, nitrocellulosestrips, membranes, sheets, duracytes, wells and walls of reaction trays,plastic tubes etc. The ligand or agent may be bound to many differentcarriers. Examples of well-known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for the purposes of the invention. Suitable methods forfixing/immobilizing said ligand are well known and include, but are notlimited to ionic, hydrophobic, covalent interactions and the like. It isalso contemplated to use “suspension arrays” as arrays according to thepresent invention (Nolan 2002, Trends Biotechnol. 20(1):9-12). In suchsuspension arrays, the carrier, e.g. a microbead or microsphere, ispresent in suspension. The array consists of different microbeads ormicrospheres, possibly labeled, carrying different ligands. Methods ofproducing such arrays, for example based on solid-phase chemistry andphoto-labile protective groups, are generally known (U.S. Pat. No.5,744,305).

The term “amount” as used herein encompasses the absolute amount of apolypeptide or peptide, the relative amount or concentration of the saidpolypeptide or peptide as well as any value or parameter whichcorrelates thereto or can be derived therefrom. Such values orparameters comprise intensity signal values from all specific physicalor chemical properties obtained from the said peptides by directmeasurements, e.g., intensity values in mass spectra or NMR spectra.Moreover, encompassed are all values or parameters which are obtained byindirect measurements specified elsewhere in this description, e.g.,response levels determined from biological read out systems in responseto the peptides or intensity signals obtained from specifically boundligands. It is to be understood that values correlating to theaforementioned amounts or parameters can also be obtained by allstandard mathematical operations. According to exemplary embodiments ofthe subject invention, the determination of an “amount” is performed bythe disclosed system, whereby a computing device determines the “amount”based on contacting and measuring steps performed by one or moreanalyzer units of said system.

The term “calculating a first ratio” or “calculating a second ratio” asreferred to herein relates to calculating a ratio of the amount ofsFlt-1 or Endoglin and the amount of PIGF by dividing the said amountsor by carrying out any other comparable mathematical calculation whichputs into a relation the amount of sFlt-1 or Endoglin towards the amountof PIGF. According to embodiments of the instant disclosure, the amountof sFlt-1 or Endoglin is divided by the amount of PIGF in order tocalculate the ratio. This calculation is carried out for the respectiveamounts determined in the said first and the said second sampleseparately yielding the first and the second ratio, respectively.

The term “comparing” as used herein encompasses comparing the firstratio to the second ratio as defined above. It is to be understood thatcomparing as used herein refers to any kind of comparison made betweenthe value calculated for the first ratio with the value calculated forthe second ratio. An increased risk for developing preeclampsia has beenfound in the studies underlying the present invention to correlate withan increase of the value of the first ratio by a factor of about 3 ormore for the value of the second ratio. The comparison referred to inthe method of the present invention may be carried out manually or by acomputing device (e.g., of a system disclosed herein).

The comparison referred to in step (c) of the method of the presentinvention may be carried out manually or computer assisted. The value ofthe ratios can be, e.g., compared to each other and the said comparisoncan be automatically carried out by a computer program executing analgorithm for the comparison. As a result of the comparison of thevalues, a slope value is obtained which indicates the factor by whichthe second ratio value differs from the first ratio value. In a furtherstep of the comparison, it is determined whether the said slope value isequal, larger or less than the factor 3. If the slope value is about 3or larger (i.e., when an increase of about 200% or more is observed), arisk for developing preeclampsia within a short period of time will bediagnosed (“rule-in”). Similarly, a slope value below factor 3 (i.e.,when an increase of less than 200%, an essentially unchanged value or adecrease is observed) shall indicate that the subject is not at risk ofdeveloping preeclampsia within a short period of time will be diagnosed(“rule-out”).

Said evaluation of the result of the comparison of the first and secondratio values can be carried out automatically as well. The computerprogram carrying out the said evaluation will provide the desiredassessment in a suitable output format. For example, a result of acomparison may be given as raw data (absolute or relative amounts), andin some cases as an indicator in the form of a word, phrase, symbol, ornumerical value which may be indicative of a particular diagnosis. Saidrule-in and/or rule-out diagnosis may be provided by the computingdevice of a system disclosed herein based on said comparison of thecalculated ratio to a first or second ratio as described herein. Forexample, a computing device of a system may provide an indicator, in theform of a word, symbol, or numerical value which is indicative of one ofa rule-in or rule-out diagnosis.

The term “about” in the context of the present invention means +/−20%,+/10%, +/−5%, +/−2% or +/−1% from the indicated parameters or values.This also takes into account usual deviations caused by measurementtechniques and the like.

Advantageously, it has been found in the studies underlying the presentinvention that a strong increase of the ratio of the amounts of sFlt-1or Endoglin and PIGF (sFlt1/PIGF or Endoglin/PIGF ratio) in a pregnantsubject which shows no or limited clinically apparent symptoms ofpreeclampsia at the time when the samples which are investigated havebeen taken is an indicator for an imminent preeclampsia, i.e., thedevelopment of preeclampsia within a short period of a few weeks, and/orimminent HELLP syndrome. Further, it has been found that thepreeclampsia developed by the said subjects being at risk is usuallypreeclampsia of the more severe early-onset-preeclampsia type. Inparticular, it was found that an increase by a factor of about 3 or moreis a reliable and predictive indicator for the aforementioned imminentpreeclampsia operating with a reasonable sensitivity and a specificityof more than 98%. A weaker increase, however, did not correlate verywell with the development of imminent preeclampsia. Remarkably, thestrong increase as specified above was predictive regardless the actualabsolute amounts or ratios of the biomarkers.

Thanks to the present invention, it is possible to more reliablydiagnose the risk for imminent preeclampsia, in particular, imminentearly-onset-preeclampsia, based on a reliable indicator which appears tobe independent on the actual absolute amounts of the aforementionedbiomarkers found in a subject. Moreover, the time consuming, expensiveand cumbersome diagnostic measures such as the current scoring systemscan be avoided when applying the method of the invention as an aid fordiagnosis. Health care management shall greatly benefit from the methodof the present invention since the need for intensive and special carerequired for pregnant females suffering from preeclampsia can be betterestimated and be taken into account for health care management purposes.

It is to be understood that the definitions and explanations of theterms made above and below apply accordingly for all embodimentsdescribed in this specification and the accompanying claims.

According to embodiments of the instant disclosure methods of thepresent invention, include preeclampsia being early-onset-preeclampsia.Accordingly, the method of the invention allows for diagnosing whether asubject is at increased risk for developing early-onset-preeclampsiawithin a short period of time, in particular if the pregnant subject isbetween week 15 and week 30 of gestation. As discussed before,early-onset-preeclampsia usually has more severe consequences thanlate-onset-preeclampsia and the subjects suffering therefrom needsupportive measures in order to ameliorate the consequences of thepreeclampsia. For example, subjects at risk can be admitted to ahospital with maternal fetal intensive care unit at an early stage.

In some embodiments of the method of the present invention, said methodfurther comprises recommending at least one supportive measure forpreeclampsia, if it is diagnosed that the subject is at increased riskfor developing preeclampsia within a short period of time.

The term “recommending” as used herein means establishing a proposal fora supportive measure or combinations thereof which could be applied tothe subject. However, it is to be understood that applying the actualtherapy, whatsoever, is not comprised by the term.

As discussed before, a subject suffering from preeclampsia requiresparticular medical care. Thus, if a subject is diagnosed to be at riskof developing preeclampsia, such a diagnosis can help to establishsuitable supportive measures for the subject in advance, i.e., beforethe preeclampsia becomes clinically apparent. According to embodimentsof the instant disclosure, said at least one supportive measure isselected from the group consisting of: close monitoring,hospitalization, administration of blood pressure reducing agents,and/or life style recommendations. With respect to the fetus,betamethasone administration may be recommended as well in order toimprove the respiratory functions in the new-born in case of a laterpremature delivery.

Embodiment of the present invention further relates to a method fordifferentiating between a pregnant subject being at risk of developingearly-onset-preeclampsia and a pregnant subject being not at risk ofdeveloping early-onset-preeclampsia comprising: a) determining theamounts of the biomarkers sFlt-1 or Endoglin and PIGF in a first and asecond sample of said subject, wherein said first sample has beenobtained prior to said second sample; b) calculating a first ratio fromsaid amounts of sFlt-1 or Endoglin and PIGF determined in the firstsample and a second ratio from said amounts of sFlt-1 or Endoglin andPIGF determined in the second sample; c) comparing the value of the saidfirst and the said second ratio, whereby a subject being at risk fordeveloping early-onset-preeclampsia is diagnosed if the value of thesecond ratio is increased compared to the value of the first ratio by afactor of at least about 3. In some cases, said early-onset-preeclampsiadevelops within a short period of time as set forth elsewhere herein indetail. According to embodiments, the said second sample has beenobtained no later than about week 30 of gestation, i.e., prior or atweek 30 of gestation.

As discussed above already, the reliable identification of subjectswhich are at risk for developing early-onset-preeclampsia is a decisivetask in health management. In particular, special care is required forpregnant females suffering from preeclampsia and, thanks to the presentinvention; the need for such care can be better estimated and be takeninto account for health care management purposes. In particular, theaforementioned method even allows identifying an early-onset type ofpreeclampsia if the second sample is obtained around week 30 ofgestation. It will be understood that preeclampsia in a subject whichexhibits no or only limited symptoms of preeclampsia until week 30 ofgestation, i.e., when the second sample has been taken, will in alllikelihood be diagnosed as non-early-onset preeclampsia since theclinically apparent preeclampsia will normally occur after week 34 ofgestation. Since the aforementioned method takes into account thedynamics of biomarkers, it allows for more reliably diagnosing theproper type of preeclampsia.

The present invention, in general, contemplates the use of thebiomarkers sFlt-1 or Endoglin and PIGF or detection agents whichspecifically bind thereto in a first and a second sample of a pregnantsubject for diagnosing whether said subject is at risk for developingpreeclampsia within a short period of time. According to embodiments ofthe instant disclosure, the biomarkers or detection agents therefore canbe used, as indicated in the aforementioned method, for diagnosingwhether said subject is at risk for developing preeclampsia within ashort period of time in a pregnant subject. According to embodiments ofthe instant disclosure, ratios of sFlt-1 or Endoglin and PIGF shall becalculated for the first and the second sample and the ratios shallsubsequently be compared to each other in order to determine the factorof increase or alteration between the two ratios, wherein an increase ofby a factor of about 3 or more is to be used as an indicator for asubject being at risk for developing preeclampsia within a short periodof time.

Moreover, the present invention also contemplates, in general, the useof the biomarkers sFlt-1 or Endoglin and PIGF or detection agents whichspecifically bind thereto in a first and a second sample of a pregnantsubject for differentiating between a pregnant subject being at risk ofdeveloping early-onset-preeclampsia and a pregnant subject being not atrisk of developing early-onset-preeclampsia.

According to embodiments of the instant disclosure, the biomarkers ordetection agents therefore can be used as indicated in theaforementioned method for differentiating between a pregnant subjectbeing at risk of developing early-onset-preeclampsia and a pregnantsubject being not at risk of developing early-onset-preeclampsia. Forexample, ratios of sFlt-1 or Endoglin and PIGF shall be calculated forthe first and the second sample and the ratios shall subsequently becompared to each other in order to determine the factor of increase oralteration between the two ratios, wherein an increase of by a factor ofabout 3 or more is to be used as an indicator for a subject being atrisk for developing early-onset-preeclampsia.

According to embodiments of the instant disclosure, a method forestablishing an aid for optimizing a risk assessment based on a clinicalprediction rule for classifying pregnant subjects is disclosed, saidmethod comprising: a) obtaining a first ratio by (i) bringing a firstsample into contact with a detection agent (detection agents) thatspecifically bind(s) to sFlt-1, Endoglin, and/or PIGF for a timesufficient to allow for the formation of a complex of the said detectionagent and the markers from the sample, (ii) measuring the amount of theformed complex(es), wherein the said amount of the formed complex(es) isproportional to the amount of the markers present in the sample, (iii)transforming the amount of the formed complex(es) into amounts of themarkers reflecting the amounts of the markers present in the sample, and(iv) calculating a first ratio from said amounts of sFlt-1 or Endoglinand PIGF determined said first sample; b) obtaining a second ratio by(i) bringing a second sample into contact with a detection agent(detection agents) that specifically bind(s) to sFlt-1, Endoglin, and/orPIGF for a time sufficient to allow for the formation of a complex ofthe said detection agent and the markers from the sample, (ii) measuringthe amount of the formed complex(es), wherein the said amount of theformed complex(es) is proportional to the amount of the markers presentin the sample, (iii) transforming the amount of the formed complex(es)into amounts of the markers reflecting the amounts of the markerspresent in the sample, and (iv) calculating a second ratio from saidamounts of sFlt-1 or Endoglin and PIGF determined said second sample; c)comparing said first ratio to a second ratio; and d) establishing an aidfor optimizing a risk assessment based on a clinical prediction rule forclassifying pregnant subjects based on the result of the comparison madein step c).

According to embodiments of the instant disclosure, a system forestablishing an aid for optimizing a risk assessment based on a clinicalprediction rule for classifying pregnant subjects is contemplated,comprising: a) an analyzer unit configured to contact, in vitro, aportion of a second sample from a pregnant subject with a ligandcomprising specific binding affinity for sFlt-1 and/or Endoglin andconfigured to contact, in vitro, a portion of a sample from a pregnantsubject with a ligand comprising specific binding affinity for PIGF, b)an analyzer unit configured to detect a signal from the portions of thesample from the subject contacted with the ligands, c) a computingdevice having a processor and in operable communication with saidanalysis units, and d) a non-transient machine readable media includinga plurality of instruction executable by a processor, the instructions,when executed, calculating an amount of sFlt-1 and/or Endoglin,calculating an amount of PIGF, calculating a second ratio from saidamounts of sFlt-1 or Endoglin and PIGF determined in the sample andcomparing the ratio thus calculated with a first ratio obtained from afirst sample, thereby optimizing the risk assessment based on theclinical prediction rule for classifying pregnant subjects.

A suitable detection agent may be, in an aspect, an antibody whichspecifically binds to the at least one marker, i.e. a detection agentwhich binds to sFlt-1, to Endoglin, or to PIGF, in a sample of a subjectto be investigated by the method of the invention. Another detectionagent that can be applied, in an aspect, may be an aptamere whichspecifically binds to the at least one marker in the sample. In yet anaspect the, sample is removed from the complex formed between thedetection agent and the at least one marker prior to the measurement ofthe amount of formed complex. Accordingly, in an aspect, the detectionagent may be immobilized on a solid support. In yet another aspect, thesample can be removed from the formed complex on the solid support byapplying a washing solution. The formed complex shall be proportional tothe amount of the at least one marker present in the sample. It will beunderstood that the specificity and/or sensitivity of the detectionagent to be applied defines the degree of proportion of at least onemarker comprised in the sample which is capable of being specificallybound. Further details on how the determination can be carried out arealso found elsewhere herein. The amount of formed complex shall betransformed into an amount of at least one marker reflecting the amountindeed present in the sample. Such an amount, in an aspect, may beessentially the amount present in the sample or may be, in anotheraspect, an amount which is a certain proportion thereof due to therelationship between the formed complex and the amount present in theoriginal sample. In yet an aspect of the aforementioned method, step a)may be carried out by an analyzer unit, in an aspect, an analyzer unitas defined elsewhere herein.

The aid for optimizing a risk assessment is established based on thecomparison carried out in step d) by allocating the subject either intoa group of subjects having an increased risk or decreased risk as setforth herein elsewhere. As discussed elsewhere herein already, theallocation of the investigated subject must not be correct in 100% ofthe investigated cases. Moreover, the groups of subjects into which theinvestigated subject is allocated are artificial groups in that they areestablished based on statistical considerations, i.e. a certainpreselected degree of likelihood based on which the method of theinvention shall operate. In an aspect of the invention, the aid foroptimizing a risk assessment is established automatically, e.g.,assisted by a computing device or the like, as described and disclosedherein.

According to embodiments of the instant disclosure, said method furthercomprises a step of recommending and/or managing the subject accordingto the result established in step d) as set forth elsewhere herein indetail, and/or adapting intensiveness of disease monitoring.

In another aspect of the invention, a system for establishing an aid foroptimizing a risk assessment based on a clinical prediction rule forclassifying subjects with pneumonia, is contemplated, comprising: a)ananalyzer unit configured to contact, in vitro, a portion of a secondsample from a pregnant subject with a ligand comprising specific bindingaffinity for sFlt-1 and/or Endoglin and configured to contact, in vitro,a portion of a sample from a pregnant subject with a ligand comprisingspecific binding affinity for PIGF, b)an analyzer unit configured todetect a signal from the portions of the sample from the subjectcontacted with the ligands, c)a computing device having a processor andin operable communication with said analysis units, and d) anon-transient machine readable media including a plurality ofinstruction executable by a processor, the instructions, when executed,calculating an amount of sFlt-1 and/or Endoglin, calculating an amountof PIGF, calculating a second ratio from said amounts of sFlt-1 orEndoglin and PIGF determined in the sample and comparing the ratio thuscalculated with a first ratio obtained from a first sample, therebyoptimizing the risk assessment based on the clinical prediction rule forclassifying pregnant subjects.

According to embodiments of the instant disclosure, a system foroptimizing a risk assessment based on a clinical prediction rule forclassifying pregnant is disclosed. Examples of systems include clinicalchemistry analyzers, coagulation chemistry analyzers, immunochemistryanalyzers, urine analyzers, nucleic acid analyzers, used to detect theresult of chemical or biological reactions or to monitor the progress ofchemical or biological reactions. More specifically, exemplary systemsof the instant disclosure may include Roche ELECSYS™ Systems and COBAS®e Immunoassay Analyzers, Abbott ARCHITECT™ and AXSYM™ Analyzers, SiemensCENTAUR™ and IMMULITE™ Analyzers, and Beckman Coulter UNICEI™ and ACESS™Analyzers, or the like.

Embodiments of the system may include one or more analyzer unitsutilized for practicing the subject disclosure. The analyzer units ofthe system disclosed herein are in operable communication with thecomputing device disclosed herein through any of a wired connection,Bluetooth, LANS, or wireless signal, as are known. Additionally,according to the instant disclosure, an analyzer unit may comprise astand-alone apparatus, or module within a larger instrument, whichperforms one or both of the detection, e.g. qualitative and/orquantitative evaluation of samples for diagnostic purpose. For example,an analyzer unit may perform or assist with the pipetting, dosing,mixing of samples and/or reagents. An analyzer unit may comprise areagent holding unit for holding reagents to perform the assays.Reagents may be arranged for example in the form of containers orcassettes containing individual reagents or group of reagents, placed inappropriate receptacles or positions within a storage compartment orconveyor. Detection reagents may also be in immobilized form on a solidsupport which are contacted with the sample. Further, an analyzer unitmay include a process and/or detection component which is optimizablefor specific analysis.

According to some embodiments, an analyzer unit may be configured foroptical detection of an analyte, for example a marker, with a sample. Anexemplary analyzer unit configured for optical detection comprises adevice configured for converting electro-magnetic energy into anelectrical signal, which includes both single and multi-element or arrayoptical detectors. According to the present disclosure, an opticaldetector is capable of monitoring an optical electro-magnetic signal andproviding an electrical outlet signal or response signal relative to abaseline signal indicative of the presence and/or concentration of ananalyte in a sample being located in an optical path. Such devices mayalso include, for example, photodiodes, including avalanche photodiodes,phototransistors, photoconductive detectors, linear sensor arrays, CCDdetectors, CMOS detectors, including CMOS array detectors,photomultipliers, and photomultiplier arrays. According to certainembodiments, an optical detector, such as a photodiode orphotomultiplier, may contain additional signal conditioning orprocessing electronics. For example, an optical detector may include atleast one pre-amplifier, electronic filter, or integrated circuit.Suitable pre-preamplifiers include, for example, integrating,transimpedance, and current gain (current mirror) preamplifiers.

Additionally, one or more analyzer unit according to the instantdisclosure may comprise a light source for emitting light. For example,a light source of an analyzer unit may consist of at least one lightemitting element (such as a light emitting diode, an electric poweredradiation source such as an incandescent lamp, an electroluminescentlamp, a gas discharge lamp, a high-intensity discharge lamp, a laser)for measuring analyte concentrations with a sample being tested or forenabling an energy transfer (for example, through florescent resonanceenergy transfer or catalyzing an enzyme).

Further, an analyzer unit of the system may include one or moreincubation units (for example, for maintaining a sample or a reagent ata specified temperature or temperature range). In some embodiments, ananalyzer unit may include a thermocycler, include a real-timethermocycler, for subjecting a sample to repeated temperature cycles andmonitoring a change in the amount of an amplification product with thesample.

Additionally, an analyzer unit of the system disclosed herein maycomprise, or be operationally connected to, a reaction vessel or cuvettefeeding unit. Exemplary feeding units include liquid processing units,such as a pipetting unit, to deliver samples and/or reagents to thereaction vessels. The pipetting unit may comprise a reusable washableneedle, e.g. a steel needle, or disposable pipette tips. The analyzerunit may further comprise one or more mixing units, for example a shakerto shake a cuvette comprising a liquid, or a mixing paddle to mixliquids in a cuvette, or reagent container.

The present invention further relates to a device adapted for diagnosingwhether a pregnant subject is at risk for developing preeclampsia withina short period of time by carrying out the aforementioned methodcomprising: a) an analyzing unit comprising a detection agent whichspecifically binds to sFlt-1 and/or Endoglin and a detection agent whichspecifically binds to PIGF, said unit being adapted for determining theamount of sFlt-1 and/or Endoglin and the amount of PIGF in a first and asecond sample of a pregnant subject; and b) an evaluation unitcomprising a data processor having implemented an algorithm for carryingout the following steps of: i) calculating a first ratio from saidamounts of sFlt-1 or Endoglin and PIGF determined in the first sampleand a second ratio from said amounts of sFlt-1 or Endoglin and PIGFdetermined in the second sample; and ii) comparing the value of the saidfirst and the said second ratio, whereby a subject being at risk fordeveloping preeclampsia within a short period of time is diagnosed ifthe value of the second ratio is increased compared to the value of thefirst ratio by a factor of at least about 3.

The term “device” as used herein relates to a system comprising theaforementioned units operatively linked to each other as to allow thediagnosis according to the methods of the invention. Exemplary detectionagents which can be used for the analyzing unit are disclosed elsewhereherein. The analyzing unit (or analyzer unit), for example, may comprisesaid detection agents in immobilized form on a solid support which is tobe contacted to the sample comprising the biomarkers the amount of whichis to be determined. Moreover, the analyzing unit can also comprise adetector which determines the amount of detection agent which isspecifically bound to the biomarker(s). The determined amount can betransmitted to the evaluation unit. Said evaluation unit comprises adata processing element, such as a computer, with an implementedalgorithm for carrying out a calculation of ratios, a comparison of saidcalculated ratios and an evaluation of the result of the comparison byimplementation of an computer based algorithm carrying out the steps ofthe method of the present invention set forth elsewhere herein indetail. The results may be given as output of parametric diagnostic rawdata. It is to be understood that these data will usually needinterpretation by the clinician. However, also envisage are expertsystem devices wherein the output comprises processed diagnostic rawdata the interpretation of which does not require a specializedclinician.

According to some embodiments of the instant disclosure, an algorithmfor carrying out a comparison between a first ratio and a second ratiodisclosed herein is embodied and performed by executing theinstructions. The results may be given as output of parametricdiagnostic raw data or as absolute or relative amounts. According tovarious embodiments of the system disclosed herein, a “diagnosis” may beprovided by the computing device of a system disclosed herein based onsaid comparison of the calculated ratios. For example, a computingdevice of a system may provide an indicator, in the form of a word,symbol, or numerical value which is indicative of a particulardiagnosis.

The present invention further relates to a device adapted fordifferentiating between a pregnant subject being at risk of developingearly-onset-preeclampsia and a pregnant subject being not at risk ofdeveloping early-onset-preeclampsia by carrying out the aforementionedmethod comprising: a) an analyzing unit comprising a detection agentwhich specifically binds to sFlt-1 and/or Endoglin and a detection agentwhich specifically binds to PIGF, said unit being adapted fordetermining the amount of sFlt-1 and/or Endoglin and the amount of PIGFin a first and a second sample of a pregnant subject; and b) anevaluation unit comprising a data processor having implemented analgorithm for carrying out the following steps of: i) calculating afirst ratio from said amounts of sFlt-1 or Endoglin and PIGF determinedin the first sample and a second ratio from said amounts of sFlt-1 orEndoglin and PIGF determined in the second sample; and ii) comparing thevalue of the said first and the said second ratio, whereby a subjectbeing at risk for developing early-onset-preeclampsia is diagnosed ifthe value of the second ratio is increased compared to the value of thefirst ratio by a factor of at least about 3.

Furthermore, encompassed by the invention is a kit adapted for carryingout the aforementioned method for diagnosing whether a pregnant subjectis at risk for developing preeclampsia within a short period of timecomprising detection agents for determining the amounts of thebiomarkers sFlt-1 or Endoglin and PIGF as well as instructions forcarrying out the said method.

The term “kit” as used herein refers to a collection of theaforementioned components, for example, provided in separately or withina single container. The container also comprises instructions forcarrying out the method of the present invention. These instructions maybe in the form of a manual or may be provided by a computer program codewhich is capable of carrying out the calculations and comparisonsreferred to in the methods of the present invention and to establish adiagnosis accordingly when implemented on a computer or a dataprocessing device. The computer program code may be provided on a datastorage medium or device such as an optical storage medium (e.g., aCompact Disc) or directly on a computer or data processing device.Moreover, the kit may, for example, comprise standard amounts for thebiomarkers as described elsewhere herein for calibration purposes.

The invention further encompasses a kit adapted for carrying out theaforementioned method for differentiating between a pregnant subjectbeing at risk of developing early-onset-preeclampsia and a pregnantsubject being not at risk of developing early-onset-preeclampsiacomprising detection agents for determining the amounts of thebiomarkers sFlt-1 or Endoglin and PIGF as well as instructions forcarrying out the said method.

The following examples, sequence listing, and figures are provided forthe purpose of demonstrating various embodiments of the instantdisclosure and aiding in an understanding of the present disclosure, thetrue scope of which is set forth in the appended claims. These examplesare not intended to, and should not be understood as, limiting the scopeor spirit of the instant disclosure in any way. It should also beunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the disclosure.

Illustrative Embodiments

The following comprises a list of illustrative embodiments according tothe instant disclosure which represent various embodiments of theinstant disclosure. These illustrative embodiments are not intended tobe exhaustive or limit the disclosure to the precise forms disclosed,but rather, these illustrative embodiments are provided to aide infurther describing the instant disclosure so that others skilled in theart may utilize their teachings.

-   1. A method for diagnosing whether a pregnant subject is at risk for    developing preeclampsia within a short period of time comprising:    -   a) determining the amounts of the biomarkers sFlt-1 or Endoglin        and PIGF in a first and a second sample of said subject, wherein        said first sample has been obtained prior to said second sample;    -   b) calculating a first ratio from said amounts of sFlt-1 or        Endoglin and PIGF determined in the first sample and a second        ratio from said amounts of sFlt-1 or Endoglin and PIGF        determined in the second sample;    -   c) comparing the value of the said first and the said second        ratio, whereby a subject being at risk for developing        preeclampsia within a short period of time is diagnosed if the        value of the second ratio is increased compared to the value of        the first ratio by a factor of at least about 3.-   2. The method of embodiment 1, wherein said first sample has been    obtained about 4 to about 5 weeks prior to the said second sample.-   3. The method of embodiments 1 or 2, wherein said pregnant subject    is between about week 15 and about week 34 of gestation, preferably,    between about week 15 and about week 30 of gestation.-   4. The method of any one of embodiments 1 to 3, wherein said short    period of time is a period of time less than 4 weeks, preferably,    between about 2 to about 3 weeks.-   5. The method of any one of embodiments 1 to 4, wherein said    preeclampsia is early-onset-preeclampsia.-   6. The method of any one of embodiments 1 to 5, wherein said method    further comprises recommending at least one supportive measure for    preeclampsia, if it is diagnosed that the subject is at increased    risk for developing preeclampsia within a short period of time.-   7. The method of embodiment 6, wherein said at least one supportive    measure is selected from the group consisting of: close monitoring,    hospitalization, administration of blood pressure reducing agents    and/or life style recommendations.-   8. A method for differentiating between a pregnant subject being at    risk of developing early-onset-preeclampsia and a pregnant subject    being not at risk of developing early-onset-preeclampsia comprising:    -   a) determining the amounts of the biomarkers sFlt-1 or Endoglin        and PIGF in a first and a second sample of said subject, wherein        said first sample has been obtained prior to said second sample;    -   b) calculating a first ratio from said amounts of sFlt-1 or        Endoglin and PIGF determined in the first sample and a second        ratio from said amounts of sFlt-1 or Endoglin and PIGF        determined in the second sample;    -   c) comparing the value of the said first and the said second        ratio, whereby a subject being at risk for developing        early-onset-preeclampsia is diagnosed if the value of the second        ratio is increased compared to the value of the first ratio by a        factor of at least about 3.    -   9. The method of embodiment 8, wherein said first sample has        been obtained about 4 to about 5 weeks prior to the said second        sample.-   10. The method of embodiment 8 or 9, wherein said pregnant subject    is between about week 15 and about week 34 of gestation, preferably,    between about week 15 and about week 30 of gestation.-   11. The method of any one of embodiments 1 to 10, wherein said first    and said second sample is a blood, serum or plasma sample.-   12. Use of the biomarkers sFlt-1 or Endoglin and PIGF or detection    agents which specifically bind thereto in a first and a second    sample of a pregnant subject for diagnosing whether said subject is    at risk for developing preeclampsia within a short period of time.-   13. Use of the biomarkers sFlt-1 or Endoglin and PIGF or detection    agents which specifically bind thereto in a first and a second    sample of a pregnant subject for differentiating between a pregnant    subject being at risk of developing early-onset-preeclampsia and a    pregnant subject being not at risk of developing    early-onset-preeclampsia.-   14. A device adapted for diagnosing whether a pregnant subject is at    risk for developing preeclampsia within a short period of time by    carrying out the method of any one of embodiments 1 to 7 and 11    comprising:    -   a) an analyzing unit comprising a detection agent which        specifically binds to sFlt-1 and/or Endoglin and a detection        agent which specifically binds to PIGF, said unit being adapted        for determining the amount of sFlt-1 and/or Endoglin and the        amount of PIGF in a first and a second sample of a pregnant        subject; and    -   b) an evaluation unit comprising a data processor having        implemented an algorithm for carrying out the following steps        of:        -   i) calculating a first ratio from said amounts of sFlt-1 or            Endoglin and PIGF determined in the first sample and a            second ratio from said amounts of sFlt-1 or Endoglin and            PIGF determined in the second sample; and        -   ii) comparing the value of the said first and the said            second ratio, whereby a subject being at risk for developing            preeclampsia within a short period of time is diagnosed if            the value of the second ratio is increased compared to the            value of the first ratio by a factor of at least about 3.-   15. A device adapted for differentiating between a pregnant subject    being at risk of developing early-onset-preeclampsia and a pregnant    subject being not at risk of developing early-onset-preeclampsia by    carrying out the method of any one of embodiments 8 to 11    comprising:    -   a) an analyzing unit comprising a detection agent which        specifically binds to sFlt-1 and/or Endoglin and a detection        agent which specifically binds to PIGF, said unit being adapted        for determining the amount of sFlt-1 and/or Endoglin and the        amount of PIGF in a first and a second sample of a pregnant        subject; and    -   b) an evaluation unit comprising a data processor having        implemented an algorithm for carrying out the following steps        of:        -   i) calculating a first ratio from said amounts of sFlt-1 or            Endoglin and PIGF determined in the first sample and a            second ratio from said amounts of sFlt-1 or Endoglin and            PIGF determined in the second sample; and        -   ii) ii) comparing the value of the said first and the said            second ratio, whereby a subject being at risk for developing            early-onset-preeclampsia is diagnosed if the value of the            second ratio is increased compared to the value of the first            ratio by a factor of at least about 3.-   16. A kit adapted for carrying out the method of any one of    embodiments 1 to 7 and 11 comprising detection agents for    determining the amounts of the biomarkers sFlt-1 or Endoglin and    PIGF as well as instructions for carrying out the said method.-   17. A kit adapted for carrying out the method of any one of    embodiments 8 to 11 comprising detection agents for determining the    amounts of the biomarkers sFlt-1 or Endoglin and PIGF as well as    instructions for carrying out the said method.

EXAMPLES Example 1: Measurement of Blood Levels of PIGF, sFLT1 andEndoglin

Blood levels of sFLT1, PIGF and Endoglin were determined using thecommercially available immunoassays. In particular; the following assayshave been used:

sFlt1 was determined with sandwich immunoassays using analysers from theRoche Elecsys™- or cobas e™-series. The assay comprises two monoclonalantibodies specific for the respective polypeptide. The first of theseantibodies is biotinylated and the second one is labelled with aTris(2,2′-bipyridyl)ruthenium(II)-complex. In a first incubation stepboth antibodies are incubated with the sample. A sandwich complexcomprising the peptide to be determined and the two different antibodiesis formed. In a next incubation step streptavidin-coated beads are addedto this complex. The beads bind to the sandwich complexes. The reactionmixture is then aspirated into a measuring cell where the beads aremagnetically captured on the surface of an electrode. The application ofa voltage then induces a chemiluminescent emission from the rutheniumcomplex which is measured by a photomultiplier. The emitted amount oflight is dependent on the amount of sandwich complexes on the electrode.The sFlt-1 test is commercially available from Roche Diagnostics GmbH,Mannheim, Germany. Further details on the assay are found in the packageinsert. The measuring range of sFlt1 includes amounts between 10 to85,000 pg/ml.

Endoglin was measured using the Quantikine™ Human Endoglin/CD105immunoassay which is commercially available from R&D Systems, Inc,Minneapolis, US. This assay employs the quantitative sandwich enzymeimmunoassay technique. A monoclonal antibody specific for Endoglin hasbeen pre-coated onto a microplate. Standards and samples are pipettedinto the wells and any Endoglin present is bound by the immobilizedantibody. After washing away any unbound substances, an enzyme-linkedmonoclonal antibody specific for Endoglin is added to the wells.Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution is added to the wells and color develops inproportion to the amount of Endoglin bound in the initial step. Thecolor development is stopped and the intensity of the color is measured.Further details on the assay are found in the package insert. Themeasuring range of Endoglin includes amounts between 0.001 ng/L to 10ng/ml.

PIGF was tested using two PIGF specific antibodies in an sandwichimmunoassay which is carried out on an ELECSYS™- or COBAS E™ seriesanalyzer (see above for details). The PIGF test is commerciallyavailable from Roche Diagnostics GmbH, Mannheim, Germany. Furtherdetails on the assay are found in the package insert. The measuringrange of PIGF includes amounts of 3 to 10,000 pg/ml.

Example 2: Analysis of the Biomarkers sFlt-1 and PIGF in OutcomePatients Which Developed Preeclampsia and in Healthy Controls

A total number of 286 patients recruited at different sites in Europewere investigated. Included in the study were pregnant women at agestational age of at least 15+0 and at most 30+0 weeks. Referencevalues for the sFlt-1/PIGF ratio are usually slightly decreasing untilabout week 28, hence a physiological increase of values in this timeinterval was not to be expected. The diagnosis at every patient visitincluded in the study was “no Preeclampsia or HELLP (PE/HELLP)” or“suspected PE/HELLP”. The diagnosis at outcome of these patients can bePE/HELLP and it was analysed here if increasing values are an indicatorfor an imminent diagnosis of PE/HELLP. Every woman contributed twovisits: her last visit before week 30+0 (visit 2) and one earlier visit(visit 1). In case of more than one options for visit 1, that one isselected, which was nearest to 3 weeks before visit 2.

Blood levels of PIGF and sFlt-1 were determined as described in Example1, above, and evaluated. The results of were as summarized in thefollowing Tables 1 to 11:

TABLE 1 Diagnosis at final outcome PE/HELLP No PE/HELLP N 37 249

TABLE 2 Gestational age at visit 1 Min. Qu.−25 Median Qu.−75 Max. MeanSD N PE/HELLP 16.14 20.00 21.71 24.57 28.00 21.83 3.43  37 No PE/HELLP15.00 20.29 21.86 24.14 28.57 21.70 3.02 249

TABLE 3 Gestational age at visit 2 Min. Qu.−25 Median Qu.−75 Max. MeanSD N PE/HELLP 20.29 26.71 27.86 28.86 30.00 27.27 2.52  37 No PE/HELLP19.14 25.57 27.14 28.71 30.00 26.78 2.41 249

TABLE 4 Days between visits Min. Qu.−25 Median Qu.−75 Max. Mean SD NPE/HELLP 14.00 28.00 34.00 47.00 90.00 38.11 17.33  37 No PE/HELLP  5.0028.00 32.00 41.00 96.00 35.53 14.40 249

TABLE 5 sFlt-1/PIGF ratio at visit 1 Min. Qu.−25 Median Qu.−75 Max. MeanSD N PE/HELLP 0.81 5.10 10.46 19.70 225.23  24.36 45.37  37 No PE/HELLP0.50 3.10  5.24  8.68 41.38  6.74  5.53 249

TABLE 6 sFlt-lPIGF ratio at visit 2 Min. Qu.−25 Median Qu.−75 Max. MeanSD N PE/HELLP 1.48 4.58 9.77 88.96 856.23  72.86 155.23   37 No PE/HELLP0.44 1.85 3.25  5.30 40.67  4.94  5.72 249

TABLE 7 Absolute change of the sFlt-1/PIGF ratio between visits Min.Qu.−25 Median Qu.−75 Max. Mean SD N PE/HELLP −17.94 −2.16 0.09 40.25742.25  48.50 131.27   37 No PE/HELLP −21.40 −3.39 −1.59  −0.36 25.60−1.81 4.61 249

TABLE 8 Percentage gain of sFlt-1/PIGF ratio Min. Qu.−25 Median Qu.−75Max. Mean SD N PE/HELLP −75.84 −36.34  5.72 245.74 1691.91  182.84372.92   37 No PE/HELLP −92.11 −51.93 −35.04  −10.63 309.43  −24.1449.77 249

TABLE 9 Listing of patients with a gain of sFlt-1/PIGF ratio of 100% ormore Ratio 1 Ratio 2 Gain Visit 1 Visit 2 PE/HELLP 10.13 20.42   101.7%25 w + 4 d 29 w + 4 d No 16.58 33.89   104.5% 26 w + 0 d 28 w + 6 d No 4.69  9.77   108.5% 20 w + 4 d 28 w + 2 d Yes 156.22  340.90    118.2%16 w + 6 d 20 w + 6 d Yes  5.82 13.06   124.2% 17 w + 1 d 30 w + 0 d Yes 8.00 18.31   128.7% 19 w + 6 d 27 w + 6 d Yes  6.28 14.39   129% 24 w +6 d 29 w + 0 d No  2.34  5.43   131.9% 23 w + 1 d 27 w + 4 d Yes  9.8523.02   133.8% 24 w + 1 d 29 w + 1 d No  3.39  8.04   137.1% 24 w + 1 d28 w + 1 d No  1.21  3.76   210.3% 24 w + 3 d 28 w + 5 d No  5.59 18.20  225.4% 24 w + 0 d 27 w + 6 d Yes 37.29 128.92    245.7% 26 w + 0 d 29w + 1 d Yes  1.71  5.95   247.2% 24 w + 3 d 28 w + 3 d No 13.62 53.87  295.5% 28 w + 0 d 30 w + 0 d Yes  8.27 33.87   309.4% 23 w + 6 d 29w + 6 d No 20.60 88.96   331.9% 25 w + 5 d 29 w + 4 d Yes 32.30 144.39   347% 25 w + 1 d 29 w + 1 d Yes 30.24 138.30    357.4% 24 w + 4 d 28w + 4 d Yes 113.98  856.23    651.2% 25 w + 1 d 27 w + 6 d Yes 19.70167.62    750.8% 24 w + 5 d 28 w + 0 d Yes 13.86 124.57    799% 20 w + 0d 27 w + 1 d Yes 20.99 239.20  1039.8% 25 w + 6 d 29 w + 6 d Yes  0.8114.50 1691.9% 21 w + 0 d 27 w + 0 d Yes

TABLE 10 Categorized gain of sFlt-1/PIGF ratio vs. final outcomePE/HELLP [%] No PE/HELLP [%] Gain <0% 17 45.9  201 80.7  Gain 0-100%  410.8   40 16.1  Gain 100-200%  5 13.5   5 2.0 Gain >200% 11 29.7   3 1.2Sum 37 100.0  249 100.0 

TABLE 11 Sens/Spec depending on gain as cutoff Cutoff at 100%Sensitivity 43.2% Specificity  96.% Cutoff at 200% Sensitivity 29.7%Specificity 98.8%

The present European PE study allows for the observation that a strongincrease of the sFlt-1/PIGF ratio (in this proposal threefold or more)seems to be a clear indicator for an imminent PE/HELLP.

Example 3: Analysis of the Biomarkers Endoglin and PIGF in OutcomePatients Which Developed Preeclampsia and in Healthy Controls

Similar patient samples as referred to in Example 2 were investigatedfor blood levels of PIGF and Endoglin (s-Eng) and evaluated. The resultswere as follows:

TABLE 12 sEng/PIGF ratio at visit 1 Min. Qu. −25 Median Qu. −75 Max.Mean SD N PE/HELLP 0.00 0.02 0.06 0.12 0.30 0.08 0.08 21 No PE/HELLP0.01 0.01 0.02 0.03 0.09 0.02 0.02 16

TABLE 13 sEng/PIGF ratio at visit 2 Min. Qu. −25 Median Qu. −75 Max.Mean SD N PE/HELLP 0.00 0.02 0.08 0.75 2.76 0.45 0.68 21 No PE/HELLP0.01 0.01 0.01 0.02 0.07 0.02 0.02 16

TABLE 14 Absolute change of the sEng/PIGF ratio between visits Min. Qu.−25 Median Qu. −75 Max. Mean SD N PE/HELLP −71.51 −19.28 117.78 641.201200.02  300.11  405.51 21 No PE/HELLP −81.01 −51.63 −37.34 −21.20372.91   1.33 119.40 16

TABLE 15 Percentage gain of sEng/PIGF ratio Min. Qu. −25 Median Qu. −75Max. Mean SD N PE/HELLP −71.51 −19.28 117.78 641.20 1200.02  300.11 405.51 21 No PE/HELLP −81.01 −51.63 −37.34 −21.20 372.91   1.33 119.4016

TABLE 16 Listing of patients with a gain of sEng/PIGF ratio of 100% ormore eratio 1 eratio2 Gain Visit 1 Visit 2 PE/HELLP 0.09 0.20  117.8% 26w + 0 d 29 w + 1 d Yes 0.01 0.02  215.5% 24 w + 3 d 28 w + 3 d No 0.100.34   236% 25 w + 5 d 29 w + 4 d Yes 0.04 0.13  245.8% 24 w + 0 d 27w + 6 d Yes 0.17 0.65  269.9% 28 w + 0 d 30 w + 0 d Yes 0.02 0.07 372.9% 23 w + 6 d 29 w + 6 d No 0.14 0.75  419.7% 25 w + 1 d 29 w + 1 dYes 0.18 1.33  641.2% 24 w + 4 d 28 w + 4 d Yes 0.10 0.81  688.1% 24 w +5 d 28 w + 0 d Yes 0.30 2.76  809.1% 25 w + 1 d 27 w + 6 d Yes 0.12 1.15 830.9% 20 w + 0 d 27 w + 1 d Yes 0.08 0.96 1051.8% 25 w + 6 d 29 w + 6d Yes 0.01 0.08   1200% 21 w + 0 d 27 w + 0 d Yes

TABLE 17 Categorized gain of sEng/PIGF ratio vs. final outcome PE/HELLP[%] No PE/HELLP [%] Gain <0%  7 33.3  14 87.5  Gain 0-100%  3 14.3   00.0 Gain 100-200%  1 4.8  0 0.0 Gain >200% 10 47.6   2 12.5  Sum 21100.0  16 100.0 

Using 100%/200% of gain as cutoff, this can be transferred in terms ofclinical sensitivity/specificity:

TABLE 18 Sens/Spec depending on gain as cutoff Cutoff at 100%Sensitivity 52.4% Specificity 87.5% Cutoff at 200% Sensitivity 47.6%Specificity  87.%

The different ratios determined for sFlt-1 and PIGF at different timepoints of gestation for patients with preeclampsia and healthy controlsare also indicated graphically in FIG. 5A. The same graph has been shownfor the Endoglin/PIGF ratios; FIG. 5B. It is apparent that the ratios ofsFlt-1/PIGF and Endoglin/PIGF show a similar distribution and, thus, aresimilar predictors for the development of preeclampsia.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within the known orcustomary practice in the art to which this disclosure pertains.

1. A method for diagnosing and treating a pregnant subject being at riskof developing early onset preeclampsia comprising: a) measuring amountsof a first biomarker selected from the group consisting of solublefms-like tyrosine kinase-1 (sFlt-1) and endoglin and a second biomarkerbeing placental growth factor (PIGF) in a first and a second sample ofsaid subject, wherein said first sample has been obtained less than 4weeks prior to said second sample, the second sample is obtained nolater than week 30 of gestation, and wherein said first and secondsample are independently selected from the group consisting of a bloodsample, a serum sample and a plasma sample; b) determining a first ratiofrom said amounts of sFlt-1 to PIGF or Endoglin to PIGF measured in thefirst sample and a second ratio from said amounts of sFlt-1 to PIGF orEndoglin to PIGF determined in the second sample; c) diagnosing thesubject as being at risk for developing early onset preeclampsia if thevalue of the second ratio is at least 3 times the value of the firstratio; and d) providing at least one supportive measure to a subjectdiagnosed as being at risk for developing preeclampsia within about 16days.
 2. The method of claim 1, wherein said pregnant subject is betweenabout week 15 and no later than week 30 of gestation.
 3. The method ofclaim 1, wherein at least one supportive measure is administration ofblood pressure reducing agents.
 4. A method for diagnosing whether apregnant subject is at risk for developing preeclampsia within a shortperiod of time comprising: a) measuring amounts of a first biomarkerselected from the group consisting of sFlt-1 and Endoglin and a secondbiomarker being PIGF in a first and second sample of said subject,wherein said first sample has been obtained less than 4 weeks prior tosaid second sample, the second sample is obtained no later than week 30of gestation, and wherein said first and second sample are independentlyselected from the group consisting of a blood sample, a serum sample anda plasma sample, wherein the amounts of first biomarker are measured by:contacting a portion of the first sample obtained from said subject withan antibody having specific binding affinity for sFlt-1 or Endoglin,thereby forming a complex between the antibody and sFlt-1 or Endoglobin,wherein the antibody having specific bind affinity for sFl-1 comprisestwo monoclonal antibodies, wherein the first monoclonal antibody isbiotinylated, and wherein the antibody having specific binding affinityfor Endoglin is a monoclonal antibody; separating the complex formed insaid step of contacting from antibody not comprising the complex; andquantifying a signal from the complex, the signal being proportional tothe concentration of sFlt-1 or Endoglin in the sample obtained from thesubject, wherein the amounts of the second biomarker are measured asfollows: contacting a portion of the first sample and the second sampleobtained from said subject with an antibody having specific bindingaffinity for PIGF, thereby forming a complex between the antibody andPIGF; separating the complex formed in said step of contacting fromantibody not comprising the complex; and quantifying a signal form thecomplex, the signal being proportional to the concentration of PIGF inthe sample obtained from the subject; b) measuring a first ratio fromsaid amounts of sFlt-1 to PIGF or Endoglin to PIGF determined in thefirst sample and a second ratio from said amounts of sFlt-1 to PIGF orEndoglin to PIGF determined in the first sample and a second ratio fromsaid amounts of sFlt-1 to PIGF or Endoglin to PIGF measured in thesecond sample; and c) diagnosing the subject as being at risk fordeveloping preeclampsia within a short period of time, wherein the shortperiod of time is about 16 days, if the value of the second ratio is atleast 3 times the value of the first ratio; and d) providing at leastone supportive measure to the subject.
 5. The method of claim 4, whereinsaid preeclampsia is early onset preeclampsia.
 6. The method of claim 4further comprising at least one supportive measure to the subject. 7.The method of claim 6, wherein at least one supportive measure isadministration of blood pressure reducing agents.
 8. The method of claim1, wherein the amounts of first biomarker are measured by: contacting aportion of the first sample obtained from said subject with an antibodyhaving specific binding affinity for sFlt-1 or Endoglin, thereby forminga complex between the antibody and sFlt-1 or Endoglobin, wherein theantibody having specific bind affinity for sFl-1 comprises twomonoclonal antibodies, wherein the first monoclonal antibody isbiotinylated, and wherein the antibody having specific binding affinityfor Endoglin is a monoclonal antibody; separating the complex formed insaid step of contacting from antibody not comprising the complex; andquantifying a signal from the complex, the signal being proportional tothe concentration of sFlt-1 or Endoglin in the sample obtained from thesubject.
 9. The method of claim 1, wherein the amounts of the secondbiomarker are measured as follows: contacting a portion of the firstsample and the second sample obtained from said subject with an antibodyhaving specific binding affinity for PIGF, thereby forming a complexbetween the antibody and PIGF; separating the complex formed in saidstep of contacting from antibody not comprising the complex; andquantifying a signal form the complex, the signal being proportional tothe concentration of PIGF in the sample obtained from the subject.